In recent years, mouse and human iPS cells have been established one after another. Yamanaka et al. induced iPS cells by transferring the Oct3/4, Sox2, Klf4 and c-Myc genes into fibroblasts from mouse and human (patent document 1 and non-patent documents 1 and 2). On the other hand, Thomson et al. group produced human iPS cells using Nanog and Lin28 in place of Klf4 and c-Myc (patent document 2 and non-patent document 3).
Various attempts have been made to enhance the iPS cell establishment efficiency. One of them is optimization of the combination of reprogramming factors. The present inventors reported that the efficiency of iPS cell establishment can be remarkably improved by using a combination of 5 factors of Oct3/4, Sox2, Klf4, L-Myc and Lin28 as reprogramming factors, and knocking down the expression of p53 by an RNAi technique (patent document 3 and non-patent document 4). Some consider that it is desirable to avoid suppression of cancer suppressor gene p53, even if transient, particularly in consideration of the application of human iPS cells to regenerative medicine, since tumorization risk should be minimized. On the other hand, Maekawa et al. reported that the efficiency of iPS cell establishment can be more remarkably improved by introducing Glis1 together with Oct3/4, Sox2 and Klf4 (OSK), into a somatic cell than by the use of 3 factors of OSK (patent document 4 and non-patent document 5). Furthermore, Maekawa et al. reported that human iPS cell is established with about 2-fold efficiency by the use of 6 factors of Oct3/4, Sox2, Klf4, L-Myc, Lin28 and Glis1 (OSKULG) than the combination of p53 shRNA with 5 factors of Oct3/4, Sox2, Klf4, L-Myc and Lin28 (OSKUL) (U.S. provisional patent application No. 61/521,153).
Viral vectors of retrovirus, lentivirus and the like have high transgene efficiency compared to nonviral vectors, and therefore, are superior vectors since they can produce iPS cell easily. However, since retrovirus and lentivirus are incorporated into the chromosome, they have safety problems in consideration of the clinical application of iPS cell. While iPS cells free of incorporation into the chromosome by using nonviral vectors such as adenovirus vector, plasmid and the like have been reported (non-patent documents 6-8), the establishment efficiency is low when compared to retrovirus and lentivirus. In addition, a stable expression strain incorporating the reprogramming factor into the chromosome is obtained at a certain frequency even when an episomal vector generally considered to resist incorporation is used, which may be due to the requested sustained high expression of reprogramming factors under selection of iPS cells (non-patent documents 7 and 9).
On the other hand, a method using an episomal vector stably and autonomously replicable outside the chromosome shows low efficiency of the above-mentioned iPS cell establishment, low frequency of spontaneous disappearance of vector due to the discontinuation of drug selection, and requires a long time (non-patent document 8). Therefore, a method of removing a vector efficiently in a short time is desired along with the improvement of iPS cell establishment efficiency. In this connection, the present inventors found an early-self-disappearing vector that falls off rapidly from the cell and already reported the vector (patent document 3, non-patent document 4 and U.S. provisional patent application No. 61/521,153).
However, a method using an episomal vector is associated with a problem of extremely low iPS cell establishment efficiency from a particular cell, for example, blood cell, as compared to a method using other vector. Since blood cell is one of the somatic cell sources extremely useful for the construction of iPS cell bank, an episomal vector method capable of establishing iPS cell efficiently, irrespective of the kind of cells, has been desired.